6 research outputs found
Switchable Elastin-Like Polypeptides that Respond to Chemical Inducers of Dimerization
Elastin-like polypeptides (ELPs)
are protein polymers that reversibly
phase separate in response to increased temperature, pressure, concentration,
ionic strength, and molecular weight. If it were possible to engineer
their phase separation to respond to specific molecular substrates,
ELP fusion proteins might be engineered as biosensors, smart biomaterials,
diagnostic imaging agents, and targeted therapies. What has been lacking
is a strategy to design ELPs to respond to specific substrates. To
address this deficiency, we report that ELP fusion proteins phase
separate in response to chemical inducers of dimerization (CID). The
rationale is that ELP phase separation depends on molecular weight,
concentration, and local hydrophobicity; therefore, processes that
affect these properties, including noncovalent dimerization, can be
tuned to produce isothermal phase separation. To test this hypothesis,
constructs were evaluated consisting of an immunophilin: human FK-506
binding protein 12 (FKBP) attached to an ELP. Under stoichiometric
binding of a CID, the fusion protein homodimerizes and triggers phase
separation. This dimerization is reversible upon saturation with excess
CID or competitive binding of a small lipophilic macrolide to FKBP.
By modulating the ELP molecular weight, phase separation was tuned
for isothermal response to CID at physiological ionic strength and
temperature (37 °C). To interpret the relationship between transition
temperature and equilibrium binding constants, an empirical mathematical
model was employed. To the best of our knowledge, this report is the
first demonstration of reversible ELP switching in response to controlled
dimerization. Due to its simplicity, this strategy may be useful to
design ELP fusion proteins that respond to specific dimeric biological
entities
Elastin-Like Peptide Amphiphiles Form Nanofibers with Tunable Length
Peptide amphiphiles (PAs) self-assemble nanostructures
with potential
applications in drug delivery and tissue engineering. Some PAs share
environmentally responsive behavior with their peptide components.
Here we report a new type of PAs biologically inspired from human
tropoelastin. Above a lower critical solution temperature (LCST),
elastin-like polypeptides (ELPs) undergo a reversible inverse phase
transition. Similar to other PAs, elastin-like PAs (ELPAs) assemble
micelles with fiber-like nanostructures. Similar to ELPs, ELPAs have
inverse phase transition behavior. Here we demonstrate control over
the ELPAs fiber length and cellular uptake. In addition, we observed
that both peptide assembly and nanofiber phase separation are accompanied
by a distinctive secondary structure attributed primarily to a type-1
β turn. We also demonstrate increased solubility of hydrophobic
paclitaxel (PAX) in the presence of ELPAs. Due to their biodegradability,
biocompatibility, and environmental responsiveness, elastin-inspired
biopolymers are an emerging platform for drug and cell delivery; furthermore,
the discovery of ELPAs may provide a new and useful approach to engineer
these materials into stimuli-responsive gels and drug carriers
Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo
Recombinant
protein–polymer scaffolds such as elastin-like
polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility,
monodispersity, and multifunctionality. We recently reported that
the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle
(FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth
in breast cancer xenografts, and reduces side effects observed with
free-drug controls. This new report significantly advances this carrier
strategy by demonstrating the coassembly of two different ELP diblock
copolymers containing drug-loading and tumor-targeting domains. A
new ELP nanoparticle (ISR) was synthesized that includes the canonical
integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in
a 1:1 molar ratio coassemble into bifunctional nanoparticles containing
both the FKBP domain for Rapa loading and the RGD ligand for integrin
binding. Coassembled nanoparticles were evaluated for bifunctionality
by performing in vitro cell-binding and drug-retention assays and
in vivo MDA-MB-468 breast tumor regression and tumor-accumulation
studies. The bifunctional nanoparticle demonstrated superior cell
target binding and similar drug retention to FSI; however, it enhanced
the formulation potency, such that tumor growth was suppressed at
a 3-fold lower dose compared to an untargeted FSI–Rapa control.
This data suggests that ELP-mediated scaffolds are useful tools for
generating multifunctional nanomedicines with potential activity in
cancer
Multimeric Disintegrin Protein Polymer Fusions That Target Tumor Vasculature
Recombinant protein therapeutics
have increased in number and frequency
since the introduction of human insulin, 25 years ago. Presently,
proteins and peptides are commonly used in the clinic. However, the
incorporation of peptides into clinically approved nanomedicines has
been limited. Reasons for this include the challenges of decorating
pharmaceutical-grade nanoparticles with proteins by a process that
is robust, scalable, and cost-effective. As an alternative to covalent
bioconjugation between a protein and nanoparticle, we report that
biologically active proteins may themselves mediate the formation
of small multimers through steric stabilization by large protein polymers.
Unlike multistep purification and bioconjugation, this approach is
completed during biosynthesis. As proof-of-principle, the disintegrin
protein called vicrostatin (VCN) was fused to an elastin-like polypeptide
(A192). A significant fraction of fusion proteins self-assembled into
multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion
proteins compete specifically for cell-surface integrins on human
umbilical vein endothelial cells (HUVECs) and two breast cancer cell
lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that,
unlike linear RGD-containing protein polymers, the disintegrin fusion
protein undergoes rapid cellular internalization. To explore their
potential clinical applications, fusion proteins were characterized
using small animal positron emission tomography (microPET). Passive
tumor accumulation was observed for control protein polymers; however,
the tumor accumulation of A192-VCN was saturable, which is consistent
with integrin-mediated binding. The fusion of a protein polymer and
disintegrin results in a higher intratumoral contrast compared to
free VCN or A192 alone. Given the diversity of disintegrin proteins
with specificity for various cell-surface integrins, disintegrin fusions
are a new source of biomaterials with potential diagnostic and therapeutic
applications
DataSheet_1_Bet v 1-displaying elastin-like polypeptide nanoparticles induce a strong humoral and weak CD4+ T-cell response against Bet v 1 in a murine immunogenicity model.docx
There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.</p